At the Marine Biological Laboratory, Vale and Sheetz worked with Bruce Schnapp and Thomas Reese.[10] They found that organelle transport occurred bidirectionally on a microtubule, not actin filament as Vale had thought, and that this movement was independent of the myosin and actin system.[11] Vale further demonstrated organelles by themselves could not move on microtubules, but movement was observed after adding the cytosol of the axon. This showed the motor protein was not present on microtubules or organelles, but dissolved in the cytosol. He also found cytosol-coated beads moved along microtubules, providing an in vitro microtubule-based motility assay.[12] In 1985, Vale, Sheetz and Reese isolated the motor protein, named it "kinesin", and showed it only moved in one direction towards the N-terminus of microtubules.[13]

Since then, Vale has switched his focus to dynein, a motor protein discovered by Ian R. Gibbons in 1965. Although its discovery occurred 20 years before kinesin, its large size hampered its isolation; research about it was relatively scarce at the time,[10] and was mainly performed by Gibbons and his team.[17] In 2006, Vale isolated dynein from yeast, and elucidated how it walked on muicrotubules.[18] He then worked with Gibbons to determine the structure of the dynein microtubule-binding domain.[19] His team also solved the structure of the dynein motor domain.[20] Vale has extended his research to other fields, including T-cell signalling[21] and RNA biology.[22]

Vale founded iBiology in 2006, a platform of free talks given by leading biologists, speaking about biological principles and their research. In 2009, he established IndiaBioscience, which holds the Yong Investigators' Meeting every year, giving young researchers in India a networking opportunity. He founded ASAPbio in 2015, promoting the use of preprints and an open and transparent peer-review process.[10]

Vale also developed Micro-Manager, a free and open-source microscopy software[23] and Microscopy 4 Kids, a website that promotes digital microscopy to children.[24]